This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Small Ras super family GTPases cycle between inactive and active conformations and play important roles in cell migration. The GTP-bound form of these GTPases interacts with effectors via Switch 1 and 2 regions, leading to their biological activities. However, the bases of the differences in biological activity of these GTPases have remained obscure and an understanding of these differences is a barrier to understanding a central node of signaling molecules that control multiple cellular processes. The simplest explanation for differing biological activities is that there might be a difference in effectors amongst Ras sub family members;however, most effectors are shared amongst them and have been identified using techniques that fail to preserve cellular localization, a key determinant of signaling output by these proteins. We have therefore set out to identify Ras GTPase interacting proteins in their native environments in mammalian cells, using tandem affinity purification (TAP) tag approach. Our initial results lead us to believe that a quantitative differential proteomic analysis of Ras effectors could lead to a major advance in understanding of the signaling properties of this important family of enzymes. The reagents and methodologies necessary for efficiently purifying the Ras and Rho proteins and thus characterizing their interactomes have been developed and validated. Thus, interacting proteins for activated Ras and Rho family proteins can readily be identified by mass spectrometric sequencing of TAP-purified complexes of activated Ras and Rho proteins. Proteins interacting with inactive mutants of Ras and Rho proteins, and proteins interacting with the TAP tag expressed alone will be subtracted from the interaction database. Specific binding to activated Ras and Rho will be evaluated by 16O/18O labeling of cellular proteins, TAP purification of Ras and Rho complexes from labeled cells, and subsequent sequence-based identification and quantification of proteins in each complex by differential mass spectrometry.